Cancer treatment mainly consists of surgery wherever possible, cytotoxic agents such as chemotherapy, and radiotherapy. Molecular therapies for cancer treatment have emerged in the last decade, such as monoclonal antibodies targeting the cell membrane receptors, and inhibitors of tyrosine kinase receptors and other kinases which target signal transduction involved in cell proliferation, death and survival. As cytostatic agents, their monotherapy regimen often lacks sufficient clinical benefit. Synergistic outcomes are often obtained by combination with cytotoxic agents but are limited by their cumulative side effects.
Most cancer treatments directly or indirectly cause DNA damage in the treated proliferating tumor cells, which ultimately leads to their death. However, several intrinsic and acquired resistances of tumors to these treatments are, at least in part, due to the tumor cells' efficient DNA repair activities. It is now well recognized that DNA repair is an important target for cancer therapy (Helleday et al. Nat. Rev. Cancer, 2008, 8:193-204). The most advanced drug development in this field is the inhibitors of PARP.
As DNA repair is an essential survival process across all living kingdom, it has multiple specialized repair pathways, and has some redundancies that make the process robust when one pathway is deficient or blocked by a therapeutic agent, such as a DNA repair inhibitor. Therefore, instead of targeting a key gene/protein involved in the DNA repair process, whatever its biological importance and clinical relevance, the innovative molecular therapy must deal with one or several key pathways as a global target, in conjunction with conventional therapies, so as to reach the most efficient cancer treatment.
It was conceived to globally target DNA lesions' sensing, signaling and repair pathways in order to disable cancers' defense to existing treatments. One strategy consists of introducing short modified DNA molecules mimicking double strand breaks (DSB), named Dbait, into cells that until then could efficiently repair DSB and thus survive. The antitumor efficacy of Dbait in association with radiotherapy (RT) or chemotherapy (CT) is explained by the fact that Dbait molecules trap the initial DSB sensing complexes, jam downstream repair signaling, subsequently disorganize all DSB repair systems (both Non-Homologous End Joining and Homologous Recombination pathways), and finally inhibit DSB repair (WO2005/040378; WO2008/034866; Quanz et al., 2009, Clinical Cancer Research 15:1308; Quanz et al., 2009, PLoS ONE 4:e6298; Dutreix et al., 2010, Mut. Res. 704:182). Ultimately, the cancer cells can no more escape their death. Dbait molecules have also been found to be effective alone, without combination with radiotherapy (RT) or chemotherapy (CT) (WO2008/084087).
However, once active agents of clinical interest are identified, the recurrent problem is to find the best way to deliver the active agents, especially for nucleic acid agents. The development and optimization of efficient non-viral DNA/RNA delivery systems has to address the toxicity issues; the “tissue and systemic barriers” such as degradation, opsonization of particles by charged serum components, rapid clearing and accumulation in non-target tissues when the active substance is administered by systemic route; and the “cellular barriers” to their delivery such as low uptake across the cytoplasmic membrane, inadequate release of DNA molecules in the active cellular compartments, and lack of nuclear targeting (required for gene therapy).
Indeed, to be effective, most of these active agents have to be taken up by the cells and to reach the cytoplasm and/or nucleus. In particular, when active agents including nucleic acids are administered in their “free” or naked form, they frequently suffer from degradation before and after uptake by target cells. Inside the cells, this degradation is mainly due to the fact that nucleic acids enter cells by endocytosis, and are sequestered in cellular endosomes that ultimately evolve into the lysosomes where chemical and enzymatic degradation is very efficient.
In the prior art, active agents have been conjugated to various carriers and have been encapsulated into liposomes, micelles and nanoparticles where they are protected from degradation in serum. The prior art also employs a variety of chemistries for covalent coupling of nucleic acids and other active agents to molecular carriers that include polymers such as dextrans or PEG or molecules aiming to decrease clearance, carriers including transferrin, and lipophilic molecules such as cholesterol linked to siRNA to enhance cellular uptake (Chen et al., 2010, J. Controlled Release 144:227). Such carriers may include targeting moieties such as antibodies, polypeptides, nucleic acids and other substances to direct the active agents to selected target cells. The prior art also discloses molecules improving endocytosis for use in pharmaceutical composition (US2008/0194540).
However, when active DNA/RNA are agents uptaken by cells through the endocytosis process, they frequently end up sequestered in endosomes where they are unable to escape, therefore greatly reducing their therapeutic potential. For instance, Zimmermann et al. showed that a cholesterol-siRNA (ApoB-1) conjugate is about 1000-fold less potent than its liposomal formulation (SNALP vector) in mice: 100 mg/kg of chol-siApoB-1 is equivalent to 0.1 mg/kg of SNALP-ApoB-1 (Zimmermann et al, Nature, 2006, 441.111-114, supplementary FIG. 1).
For nucleic acids, the prior art has tried to solve this problem through the use of cationic polymers such as polyethylenimine (PEI) (WO96/02655), liposomes with fusogenic lipids or peptides such as SNALP vector. PEI is able to destabilize endosomes by a well-described proton sponge effect and therefore facilitate the release of the nucleic acid. However, the use of PEI is often limited by its cytotoxicity and so far has not been approved for use in humans. Liposomal formulation also exhibits toxicity and limited encapsulation of nucleic acids (usually in the range of 1-2 mg/mL) that may not be suitable for the application that requires high payload of nucleic acid agents.
“Endosomolytic” agents such as chloroquines are known to enhance the transfection of nucleic acids by facilitating their escape from endosomes into the cytoplasma in cultured cells. However, the use of chloroquine is limited to in vitro use and has only rarely been evaluated for assisting nucleic acid delivery in vivo. This may be due to reports in the art of nucleic acids that teach away from its in vivo use due to chloroquine toxicity.
Benns, et al. (2000, Bioconj. Chem 11: 637) reported that “Although chloroquine has proven to aid in the release of the plasmid DNA into the cytoplasm, it has been found to be toxic and thus cannot be used in vivo” This problem is partly due to the fact that relatively high concentrations of free chloroquine are needed to reach the same site as the nucleic acid (i.e., plasmid DNA) in the endosome. Similarly, Zhang et al. (2003, J Gene Med 5:209) studied in vivo use of chloroquine for gene delivery to the liver. In this article, they used a plasmid together with a peptide (polylysine/molossin) as a DNA vector. They concluded that, despite chloroquine being effective for promoting gene delivery to the liver, multiple dosing is required and its use is limited by systemic toxicity. Indeed, they demonstrated that the acute systemic chloroquine toxicity limits in vivo use to levels which are substantially below those required for optimal gene delivery. Local delivery of chloroquine is also limited by local toxicity of chloroquine and by its diffusion away from the site of delivery. Finally, they did not observe a gene delivery, or a very low level, when naked DNA is used.
In this context, WO02007/040469 discloses that the solution to the problem of the high necessary concentration of chloroquine may be overcome by covalently coupling the chloroquine to the active agent, thereby reducing the overall dosage needed. WO2009/126933 proposes to covalently link the nucleic acid to deliver both to an endosomolytic agent and to a targeting ligand.
Chloroquine and its derivatives such as hydroxychloroquine are used in curative and prophylactic treatment of malaria. It has also been studied for use in combination with radiotherapy and/or chemotherapy of cancers (Sotelo et al., 2006, Ann Intern Med 144:337-342; NCT01023477 and NCT00969306). The hypothesis is that chloroquine/hydroxychloroquine inhibits autophagy, which is a normal cell defense process, by exporting therapeutic agents to lysosomes where they are degraded.
In conclusion, optimization of nucleic acid-based therapies requires further addressing the efficiency and cytotoxicity of synthetic DNA delivery systems.